Vibrio cholerae causes the fatal epidemic diarrheal disease cholera. The expression of its primary virulence factors, toxin-coregulated pilus and cholera toxin, occurs via a transcriptional cascade involving several activator proteins and serves as a paradigm for the regulation of bacterial virulence. ToxT, an AraC-type regulator, directly activates the promoters of the primary virulence factors. The expression of toxT requires cooperation between two homologous pairs of transmembrane activators, ToxRS and TcpPH. Activation of tcpPH expression initiates the cascade by a unique interaction between two regulatory proteins AphA and AphB. AphA is a member of a new winged-helix transcriptional regulator family and AphB is a LysR-type activator. Transcriptional activation at these various promoter occurs only in response to certain environmental stimuli. The long term goals of this proposal are to understand the molecular basis of this regulation so as to facilitate the development of better strategies to control the infectivity of V. cholerae. Achieving these goals requires an understanding of the molecular mechanisms by which the regulatory proteins function at their cognate promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. One such stimulus, cell density, influences the cascade through the quorum sensing regulator HapR which represses the expression of the aphA promoter. Aim 1 focuses on elucidating the mechanism by which HapR represses aphA expression. Evidence indicates this process involves antagonizing the function of two distinct activators, Lrp, the leucine responsive regulatory protein, and VpsR, the biofilm regulator. Aim 2 sheds new light on the molecular mechanisms by which pH influences the expression of the cascade by the recent discovery that AphB plays a role in the acid tolerance response in V. cholerae and regulates cadC and a number of other genes potentially involved in this response. Elucidating the relationship between AphB and the regulation of acid tolerance will provide new insights into how the activity of the protein is influenced by environmental stimuli. Aim 3 focuses on investigating the unique cooperative mechanism between AphA and AphB that initiates virulence gene expression in V. cholerae. This proposed work will facilitate future efforts to identify new molecules that interfere with the functions of these regulators and which may serve as novel antivirulence drugs.